In vivo evidence implicates uncoupling proteins UCP2 and UCP3 in the etiology of type 2 diabetes, in the mitigation of metabolic syndrome, and in reducing cellular damage due to reactive oxygen species. These functions are consistent with an uncoupling role for UCP2 and 3; however data in the literature are mixed in confirming such a role. Moreover, information on regulation of UCP2 and 3 function by nucleotides and fatty acids is lacking. These problems require a thorough biochemical approach, which will be applied in this project by measuring proton flux catalyzed by the purified, recombinant UCP1, 2 and 3 and by measuring proton flux in mitochondria isolated from cells expressing high amounts of UCP2 and 3. The specific aims are (1) To identify biological compounds that activate UCP-mediated uncoupling; (2) To determine whether native UCP2 and 3 uncouple isolated mitochondria and to reconstitute native UCP2 and 3 activity to validate the properties of the recombinant proteins; (3) To determine inhibitory affinities of UCP1, 2 and 3 for nucleotides and acyl CoA esters; (4) To investigate the relative roles of saturated and unsaturated fatty acids in the transport mechanisms of UCP1, 2 and 3. Studies on the purified proteins will use protocols established in the laboratory for expression, extraction, purification, reconstitution, and assay of UCP1, 2 and 3 function in liposomes. UCP1 is included in these studies as a gold standard for the validity of these techniques; it is the only UCP for which native reconstitutive activity is presently available. Studies on isolated mitochondria will use tissues known to express UCP in high amounts, such as rat lung. Triiodothyronine will be used to stimulate UCP3 expression in rat skeletal muscle, and lipopolysaccharide will be used to stimulate UCP2 expression in spleen and liver. These studies will provide new understanding of the mechanisms of UCP2 and 3 function and regulation and will also resolve mechanistic questions that have been raised about these proteins.